1.1 General Introduction
Many genes exist as multiple alleles which differ from each other by small differences in sequence. Individuals are often heterozygous with respect to the alleles of particular genes; i.e. individuals often have two different alleles of the same gene.
In some circumstances, it is desirable to separate the alleles of a gene from a mixture of the alleles. For example, when it is desired to carry out a test to determine which alleles of a gene are carried by a heterozygous individual, it is often necessary to separate the two alleles before carrying out the test because the presence of two alleles in one test can prevent meaningful results from being obtained.
In view of the fact that the difference between the alleles of a gene can be as little as one nucleotide, it is often difficult to separate the alleles from a mixture of the alleles. These difficulties are increased in genes which have a large number of different alleles, such as the major histocompatibility complex (MHC) genes (e.g. the human leucocyte antigen (HLA) class I and II genes which have over 500 known alleles).
Identity of alleles is frequently essential in a clinical setting, for example, HLA matching between a bone marrow or kidney recipient and donor is one of the major factors influencing transplant success. Up to date the most favourable bone marrow transplant (BMT) and kidney transplant results have been obtained using sibling donors who are genotypically HLA-identical to the recipient but such donors are available for only about 30% of patients(1-5). BMT using unrelated donors can be successful, but these transplants have higher rates of graft failure, increased incidence and severity of Graft versus Host Disease and more frequent complications related to delayed or inadequate immune reconstitution (4).
New molecular biological methods for detection of genetic polymorphism currently provide an opportunity to improve e.g. HLA matching of unrelated donors as well as a research tool to investigate the relationship between disparity and transplant complications. These molecular typing methods include sequence-specific amplification, hybridisation with oligonucleotide probes, heteroduplex analysis, single strand conformation polymorphism and direct nucleotide sequencing.
Each of these molecular approaches has been used for routine HLA class II typing (6), but a variety of reasons related to the HLA class I gene structure has complicated their application to class I typing. The reasons for these limitations are the extensive polymorphism of each class I locus and the degree of sequence homology between the loci. In addition, sequence homology between class I classical and non-classical genes and the reported 12 pseudo genes can cause problems for specific locus amplification(7).
The low extent of “allele specific” sequences at polymorphic sites is a feature of the HLA class I genes that has limited the resolution of all current DNA typing approaches. An “allele specific” sequence is a sequence that is only present in one allele and can therefore be used to distinguish the allele from other alleles.
The main problem which complicates the identification of an allele is the presence of a mixture of alleles, as well as contamination by segments of DNA which have homology to the allele it is wished to identify and which are co-amplified in PCR. Current typing methods are sometimes unable to resolve the allele it is wished to identify from contaminating DNA fragments. Separation techniques such as single strand conformation polymorphism (SSCP) can only partially resolve this problem.
Methods for Allele Separation
1.2 Sequence Specific Primer Amplification (PCR-SSP)
This method utilises both the group-specific and, when present, allele-specific sequence sites in PCR primer design. The SSP design is based on the amplification refractory mutation system (ARMS), in which a mismatch at the 3′ residue of the primer inhibits non-specific amplification(8.9).
Although each SSP reaction may not individually provide sufficient specifity to define an allele, the use of combinations of sequence specific primers allows the amplification of their common sequences to give the desired HLA specificity.
However, despite its high accuracy, PCR-SSP is only in some cases more informative than serology. The reason for this is the low occurrence of allele specific sequence motifs in the exons and this limitation has stimulated a vast amount of research into the identification of allele specific motifs even in the intron sequences(10). However, up to date this approach has not contributed considerably to the identification of more alleles.
Another limitation of this method is that it detects a limited number of polymorphic sequences which are utilised to predict the entire sequence. If an unknown allele is present in a particular sample this extrapolation may be incorrect.
In addition, the successful use of the technique relies on group specific amplification and therefore prior knowledge of broad HLA specificity is needed.
1.3 Single Strand Conformation Polymorphism (SSCP)
This technique is based on the electrophoretic mobility of single stranded nucleic acids in a non-denaturing polyacrylamide gel, which depends mainly on sequence-related conformation(11-13). The technique can be employed for isolating single alleles which could then be used for further manipulation and analysis such as direct sequencing. The pattern of bands obtained after electrophoresis may be diagnostic for an allele(14,15).
The major disadvantage of SSCP is the tendency of DNA single strand to adopt many conformational forms under the same electrophoretic conditions resulting in the presence of several bands from the product; this makes the identification more difficult. In addition there is a high degree of variation and inconsistency in the sensitivity of this method for detecting mutations or allelic variations and there is a physical limitation in the size of the DNA fragment which is of the order of 200-400 base pairs(16).
1.4 Denaturing Gradient Gel Electrophoresis (DGGE) and Temperature Gradient Gel Electrophoresis (TGGE) (17,18) 
The underlying principle of both techniques is the difference in the degress of melting between two alleles (double stranded DNA) which results in a reduction of mobility of the DNA fragments in polyacrylamide gels containing a denaturing reagent (DGGE) or a temperature gradient (TGGE).
Both techniques have been used frequently for screening mutations in genetic systems with one or two variants. They are only rarely used for the separation of alleles in highly polymorphic systems such as HLA.
Both techniques require specific conditions for a particular system under investigation and, in addition, where two alleles share common sequence segments with low melting points they may not always be differentiated. The simultaneous melting of both alleles will produce very similar retardations.
1.5 Cloning of DNA
This is the classical method of preparation of a single sequence, i.e. the sequence derived from a single allele. A variety of constructs has been used to introduce the required DNA fragment into a plasmid and grow sufficient copies for analysis. This method yields pure samples of the analyte, but is time consuming to perform and several clones are normally tested to ascertain the homogeneity of the product.
Methods for the Identification of Alleles
1.6 Heteroduplex Analysis
Fully matched DNA duplexes are more stable than those with base mismatches. Instability of the duplex increases with the number of nucleotide mismatches; these cause formation of loops and bends in the linear DNA fragment which produce an increasing “drag effect” in polyacrylamide gels which retard the affected migrating bands(19-21).
Mismatched DNA hybrids (heteroduplex) may be formed at the end of each PCR cycle between coamplified alleles from a particular locus or loci due to primer cross reaction at sites with similar sequences. During the annealing stage of each cycle of the PCR, a proportion of sense strands of each allele may anneal to anti-sense strands of different alleles. The banding pattern obtained in PAGE analysis can be useful for identifying the alleles involved in the reaction(22-24).
Heteroduplex analysis is an approach that has been utilised to compare HLA genes of a particular donor and recipient. HLA genes are amplified, denatured (melted into single strands) and mixed together under conditions that promote renaturation to form double stranded molecules. If the HLA genes of a donor and recipient are similar but not identical, heteroduplexes will form consisting of one strand of an allele of donor origin and a second strand from a different allele of recipient origin(25,26). The sensitivity of this method can be increased by adding DNA from an HLA allele that is not present in the donor or recipient.
The major advantage of heteroduplex analysis is that it is relatively easy and inexpensive. Limitations of this approach include inability to detect certain HLA disparities, potential detection of irrelevant silent mutations and lack of specific information regarding the nature of the alleles involved.
Up to date this approach has been used for HLA class II typing with limited success. Its application to class I typing has not been successful.
1.7 Sequence Specific Oligonucleotide Probes (PCR-SSO)
SSO typing involves amplification of HLA alleles from a particular locus followed by hybridisation with a panel of oligonucleotide probes to detect polymorphic sequences that distinguish one allele or group of alleles from all others. In polymorphic systems a one step operation may not always differentiate all the known alleles; selected primers can be used to achieve amplification of individual alleles which are then identified by specific probes. This second stage of oligotyping is often referred to as high resolution oligotyping(6).
The advantages of the PCR-SSO method are specificity, sensititivity, simplicity, reproducibility, and it is relatively inexpensive to operate and allows simultaneous processing of many samples. This approach has been applied successfully, for example, to typing of HLA class II alleles.
The major methodological drawback of this approach is that the complexity of the technique is directly related to the number of alleles under investigation and the presence of two alleles in the heterozygous condition can complicate the identification process.
Publishing oligotyping methods could result in incorrect interpretation of data if certain combinations of recently discovered alleles are present in a specimen. It is therefore necessary to update the reagents used in the identification step. Several typing approaches for HLA-A and B based on PCR-SSO have been published; these typically require over 40 and 90 probes respectively(27,28). The operation of these methods is time consuming and the resolution obtained is only moderate.
1.8 Nucleotide Sequencing
DNA templates for sequencing can be produced by a variety of methods, the most popular being the sequencing of cloned genomic or cDNA fragments, or the direct sequencing of DNA fragments produced solely by PCR (as in 1.2 above). These templates represent a single sequence derived from one haplotype. Alleles from both haplotypes of a heterozygous sample may be co-amplified and sequenced together using locus-specific PCR primer.
The recent availability of computer software, which allows the user to align the derived sequence against established sequence libraries, has facilitated the analysis and allele assignments for heterozygous samples in which both a templates are sequenced at the same time(27). The effectiveness of this method depends on the amount and frequency of ambiguous heterozygous combinations, for example there are many HLA class II alleles that when present together in one sample cannot be differentiated by this method. The number of such ambiguous combinations of allele sequences is even greater for HLA class I alleles.
Up to date two HLA class I typing approaches based on direct sequencing have been published. Both require serology information followed by allele specific PCR amplification and then direct sequencing(14,30). More recent practice, however, is to amplify DNA fragments without prior knowledge of the allele groups and to use locus specific PCR amplification. Theoretically these approaches should give the highest resolution, but they are beset by ambiguous sequence combinations which cannot be resolved satisfactorily and in practice these methods are expensive and difficult to perform routinely.